The proposed work involves the chemical characterization of enzyme intermediates involved in peroxidative enzymatic halogenation reactions catalyzed by chloroperoxidase. Compound I, Compound II and an enzyme-bound oxidized halogen species are of immediate concern because all three represent oxidized intermediates in chloroperoxidase reactions. We also plan to continue our work on the characterization of the heme ligand structure of native chloroperoxidase. Previous work has established a close relationship between cytochromes of the P-450 type, model compounds containing a thiolate anion as an axial ligand to the heme iron and native chloroperoxidase. However, direct analyses of crystalline chloroperoxidase preparation fails to detect the presence of free sulfhydryl groups in the native enzyme. We shall also continue our work on the lipid activation of pyruvate oxidase. This enzyme can be activated 25 to 500 fold by binding lipid amphiphiles to a unique high affinity lipid binding site on the enzyme. We are approaching the identification of this site through the development of covalent affinity labels for the lipid binding site.